Gene Cloning

360 Gene Cloning
MS
503 750 1400 1650 2550
Mass
Collision
chamber
MS
57 117 186 230 273 317 386 446
N
N
N
N
117
113
87
129
C
N-Val Leu Ser Glu Gly-C
Figure 11.24 Protein sequencing by MS/MS. A protein is first digested with
trypsin and the tryptic fragments separated by MS. One of the fragments is
selected during the first MS and passed through a “collision” chamber which
fragments the peptide into two populations of fragments. One set have a
common N terminus and vary in size by one amino acid, whereas the second
have a common C terminus and also vary in size by one amino acid. The mass of
each fragment is determined by a second MS procedure. The N-terminal and C-
terminal fragments can be differentiated. The fragments can be arranged by size
giving two ladders, the N-terminal (blue) and the C-terminal (gray). The
difference in size between the two adjacent fragments indicates the identity of
the residue.
one end of the original proteolytic fragment, the individual “rungs” of the
ladder representing the mass of one peptide fragment, and the difference
in mass between two adjacent fragments is determined by which amino
acid is present at that position. It is therefore relatively straightforward to
determine the amino acid sequence of each proteolytic fragment. Each